Lentivirus-based gene transfer systems represent a promising gene delivery technology, as they integrate into the genome of the target cell and mediate sustained expression of the transferred gene. To date, the majority of studies have used vectors based on the Human Immunodeficiency Virus (HIV), a human pathogen and the causative agent of AIDS. Advanced Vision Therapies, Inc. (AVT) has developed a proprietary lentiviral vector system based on the bovine immunodeficiency virus (BIV), an animal lentivirus not associated with human disease. The BIV vectors combine the transduction efficiency of the HIV-based vectors with the safety advantages of animal-based lentiviral vector systems. The BIV vectors are capable of transducing a variety of non-dividing cells in vitro, and retinal, neuronal, and spleen cells in vivo, leading to sustained transgene expression. Importantly, BlV-vector mediated delivery of an anti-angiogenic transgene efficiently blocked retinal neovascularization in a relevant rodent model, suggesting that the AVT vector is suitable for clinical applications. A limitation to the use of integrating vectors is the potential for variations in gene expression associated with the chromosomal insertion site and the potential for disruption of normal gene function caused by integration site selection. Indeed, insertional mutagenesis has recently become a reality following the discovery of three gene therapy-related leukemias in children treated for the fatal disease, X-linked SCID. Insertion site mapping studies using HIV-based vectors have found that while insertion is a non-specific event, it is nonrandom. Currently, a lentiviral vector capable of efficient, site- specific integration does not exist. Such a system would have immediate clinical utility. Here, we propose to develop and evaluate a novel system to mediate directed BIV integration. To achieve site-specific integration, we propose to fuse a modified BIV integrase lacking its native cellular DNA binding activity to a site-specific DNA binding protein with the intent of directing integration to specific target site. There are three specific aims to this pivotal project. Specific Aim 1. Identification of BIV integrase mutants that lack celluar DNA binding activity. A series of integrase mutants will be generated and evaluated for lack of non-specific DNA binding activity in a novel in vitro assay. Specific Aim 2. Construction of modified BIV integrase-DNA binding fusion proteins. The most promising integrase mutants from Specific Aim 1 will be fused to a novel DNA binding protein and evaluated for integrase and specific DNA binding functions in vitro. Specific Aim 3. Incorporation of the integrase fusion protein into the BIV vectors and evaluation of site-specific integration. Vectors encoding the modified integrase will be generated and vector insertion sites mapped and compared to those of the unmodified vector. Phase II studies will focus on the evaluation of site-specific integration and vector efficacy in rodent models of ocular neovascularization. [unreadable] [unreadable] [unreadable] [unreadable]